Method for producing l-arginine by fermentation

ABSTRACT

L-Arginine is produced by fermenting a culture medium containing n-alkanes as a carbon source by mutants of Brevibacterium and Arthrobacter capable of growing on a medium containing more than 200 Mu g/ml sulfaguanidine or 1,000 Mu g/ml 2-thiazolealanine.

United States Patent 1 Int. Cl. C12d 13/06 Kubota et al. Apr. 2, 1974 METHOD FOR PRODUCING L-ARGININE [58] Field of Search 195/28 R, 29

BY FERMENTATION [75] Inventors: Koji Kubota, Kanagawa; Takiko [56] References Cited Onoda, Tokyo; Hirotaka Kamijo; Fumihiro Yoshinaga, both of 3 222 258 12 1965 r k l 1 Kanagawa; Shinji Okumura y I rzu a eta. 195/3 H X all of Japan l u Primary ExaminerLi0nel M. Shapiro [73] Assrgnee: AjlllOll'lOtO Co., Inc., Tokyo, Japan Assistant Examiner R' penlend 22 Filed: June 19, 1972 21] Appl. No.: 264,316 [57] ABSTRACT L-Arginine is produced by fermenting a culture medium containing n-alkanes as a carbon source by mut v [3O] F'orelgn Apphcatlon i Data tants of Brevibacterium and Arthrobacter capable of .iUllC 21, 1971 Japan 46-44620 g g on a containing more than 1,000 l2-th' l l 52 US. Cl 195/28 R Sulfaguamdme 3 Claims, N0 Drawings sulfaguanidine or METHOD FOR PRODUCING L-ARGININE BY FERMENTATION This invention relates to a method for producing-L- arginine (hereinafter referred to as arginine) by fermentation.

Arginine is an important amino acid widely used for medicine and feed, and has been prepared on a commercial scale from protein hydrolyzates at relatively high cost in a complex isolation procedure. It is produced by certain wild strains of Corynebacterium and Brevibacterium produce arginine from hydrocarbons in a very low concentration (US. Pat. Nos. 3,222,258 and It have now been found that certain mutant strains of microorganisms belonging-to genus Brevibacterium or Arthrobacter and capable of assimilating n-alkanes produce significant amounts of arginine in an aqueous medium containing n-alkanes as the carbon source.

The mutant strains employed in the process of this invention can be readily derived by known mutation methods from parent strains of genera Brevibacterium and Arthrobacter capable of assimilating n-alka'nes. The parent strains are incapable of producing extracellular arginine.

Usually, an effective arginine producing mutant 7 strain is found to be resistant to a growth inhibiting agent for theparent strain, such as Z-thienylserine, D- serine, ethionine, 2-thiazole'al'anine, 'a amino-B- hydroxyvaleric acid, 6-chloropurine and sulfa drugs (sulfaguanidine, sulfamerazine, sulfisomezole, s'ulfisoxgrowing on a medium containingmore than. 200 ug/ml more than 1,000 pg/ml; 2- thiazolealanine is most effective. Sometimes the addition of characteristic nutrients required for growth of the resistant strain improves the yield of arginine.

The most effective arginine producing mutants found so far are Brevibacterium ketoglutamicum AJ-3351 (PERM-P 974) and Arthrobacter paraffineus AJ-3352 (PERM-P 975). The PERM-P -number is the deposit accession number of the Fermentation Research Institute, Agency of lndustrialScience and Technology, the Ministry of International Trade and Industry, of No. l-8-5, lnagehigashi, Chiba-shi, Chiba-ken, Japan.

The culture media used in the present invention are conventional in themselves, and contain n-alkanes'as the assimilable carbon source, assimilable nitrogen sources, inorganic ions and, if necessary, minor organic nutrients. Assimilable nalkanes have nine to twenty carbons. The n-alkanes are added to aqueous media in an amount of l' to 15 g/dl. Assimilable nitrogen sources include ammonium salts, nitrate salts, amino acids, urea and gaseous ammonia. For a good yield of arginine, the fermentation is carried outaerobically with aeration and agitation. Best yields require pH control within the range of to 9. The desired pH may be maintained by means of gaseous or aqueous ammonia, calcium carbonate, alkalimetal hydroxide, urea, and-organic or inorganicacids.

When the fermentation is carried out at 2437C,

Example 1 20 Ml batches of a medium containing g/dl nalkanes (a commercial product containing n-alkanes '-azole, etc.). Especially, a mutant strain capable of havingtwelve-to eighteen carbon atoms), 2 g/dl (NH,,) HPO 0.1 g/dl KH PQ, 0.1 g/dl K HPO 0.1 g/dl MgSO -7H O, l mg/dl FesOfll-l O, l mg/dl MnSO -4H' O, 30 pg/l biotin, 2 mg/l thiamine-HC], 0.5 ml/dl soybean protein hydrolyzate, 0.1 ml/dl corn steepliquor and 2 g/dl CaCO and of pH 7.0 were placed in 500 m1 flasks, and were sterilized by heat. Brevibacterium ketoglutamicum AJ 3351 (PERM-P 974) previously cultured at 30C for 24 hours on a medium con-' taining 1 g/dl peptone, l g/dl yeast extract, 0.5 g/dl NaCl and 2 g/dl agar was transferred to the flask and cultured for 72 hours at 31C with shaking. The cultured broth was found to contain 2.4 g/l L-arginine.

One liter of the cultured broth was centrifuged to remove cells. The supernatant was passed through a column packed with a cation exchange resin (Ambe'rlite C-SO NH, -form), and arginine was eluted with 2N NH OH. The eluate was concentrated to precipitate crude crystalline arginine. 12 G of the crystalline material obtained- Example 2 Arthrobacter paraffineus AJ 3352 was cultured by a method analogous to that ofExample 1, and 1.5 'g/l arginine was accumulated in the medium.

What is claimed is: y

l. A method of producing L-arginine which comprises c ulturingan L-arginine-producing mutant strain of Brevibacterium or Arthrobacter and capable of assimilating n-alkanes, in an aqueous medium containing n-alkanes as the carbon source, an assimilable source tant strain is Arthrobacter paraffineus PERM-P 975 

2. A method as set forth in claim 1, wherein said mutant strain is Brevibacterium ketoglutamicum FERM-P
 974. 3. A method as set forth in claim 1, wherein said mutant strain is Arthrobacter paraffineus FERM-P
 975. 